The present invention relates to the field of monoclonal antibodies and their use in therapeutic applications. More particularly, bispecific antibodies have binding specificity for a prostate tissue marker and also binding specificity for CD3 antigen present on the surface of T lymphocytes.
Prostatic cancer is a highly intractable disease which affects an increasing number of men, and now has surpassed lung cancer as the most frequently diagnosed cancer in men in the United States. Prostatic cancer is often curable if the tumor is confined to a small region of the gland and found at an early stage, and is destroyed by radiation or surgically removed in its entirety. Unfortunately, a great many prostatic cancers have already infiltrated surrounding tissue or have metastasized to remote sites, frequently the bone marrow, before initial detection. In this instance, radiation and other therapies may be more palliative than curative.
Once it was recognized how androgen dependent both normal development of prostatic cells and tumor growth were, androgen deprivation therapy appeared to offer a systemic mode of treatment. In fact, a dramatic remission in symptoms and tumor regression are observed in instances of castration or hormonal drug suppression, for which Charles Huggins won the Nobel Prize in 1966. However, the effects of androgen deprivation were temporary, and the cancers soon relapse with emergence of a new androgen independent cell type, which is unusually aggressive and resistant to all treatment modalities. In such instances, it is only the relatively slow progression of the disease in older patients and the intervention of other morbid processes, that permitted such patients to escape the inevitable outcome of the cancer. For a general review of issues in conventional treatment of prostate cancer, see xe2x80x9cProstate Cancer Working Groupxe2x80x9d, Coffey, D. S., Chair, Cancer Res., 51: 1498 (1991).
Androgen deprivation is a useful therapy. Among the androgen suppressive drugs licensed by the FDA, are Stilphostrol, which is a phosphorylated nonsteroidal estrogen, and Lupron, a synthetic nonapeptide analog of GnRH. The relapse of temporary remission and emergence of androgen independent malignant cells is variable, and hormonal therapy offers many patients sustained relief for a time. It is not understood how the tumor cells overcome their androgen dependence, or if they do, since it has not been ruled out that, at least in some instances, the androgen independent cells arise from a different precancerous clone.
Nevertheless, new treatments are clearly needed. In the last few years, there has been a significant trend towards detection of prostate cancer more frequently and at earlier stages, brought about largely by the availability of more sensitive and accurate tests for the quantitation of Prostate Specific Antigen (PSA). Concomitantly, more is known about the molecular biology of prostatic tumor expression, although how these molecular discoveries can be translated into effective therapies is not clear. For example, the androgen dependence of the early malignant cells focusses attention on the AR gene and the SRD5A2 gene. Abnormal gene expression can result from gene amplification (AR) leading to altered expression, and from genetic polymorphisms (SDR5A2) associated with high risk individuals.
Karp, et al., Cancer Res., 56: 5547 (1996) describes a number of potential therapies based on some of the molecular observations. For example, targeting antiandrogen/antiestrogen mechanisms with inhibitors of steroid 5alpha-reductase (Finansteride, etc., or steroid aromatise (Exemestane, etc.) may offer therapeutic possibilities. Also, drugs that suppress cellular proliferation, such as E-Cadherin mimetics, PD 153035 (through EGF receptor interaction), or Fluasterone (inhibition of nucleotide synthesis), may be effective where the molecular abnormality in a given tumor is diagnosed.
Monoclonal antibodies have been used in oncology as both diagnostic tools and therapeutic adjuncts. Unconjugated antibodies have been used in patients with acute leukemias (see more recent review). An antiganglioside monoclonal antibody has been used in some patients with melanomas to some advantage (see Houghton, et al., 85: 1242 (1985)). For conjugated antibodies, monoclonals bearing radionuclides have been evaluated. 131I and 90Y have been most extensively studied, although these are associated with some toxicity. Other antibody conjugates include biologic toxins (such as ricin, S. exotoxin, etc.) and chemotherapeutic drugs (such as methotrexate, or Doxorubicin).
A number of prostate-reactive antibodies have been produced and evaluated to various degrees. The most extensively studied antibody is designated 7E11 and reacts with PSMA. Radiolabelled 7E11 reacts specifically with prostate cancer cells, and has proven useful as a diagnostic agent in patients with occult recurrence of prostate cancer, as described in Kahn, et al., J. Urol., 152: 1490 (1994), and elsewhere. The use of this antibody (7E11-C5.3) in phase I clinical trials, labelled with 111In, is discussed in Monoclonal Antibodies 2: Applications in Clinical Oncology, ed. A. A. Epenetos, Chap. 32, Chapman and Hall Medical: 1993.
This antibody, however, has a number of limitations. It recognizes an intracellular rather than a surface epitope, thus having limited value in targeting viable cells. Thus, there exists a great need for monoclonal antibodies which have the desired specificity, and target epitopes of stably expressed antigen molecules on the surface of the target tumor cell.
The immune mechanisms leading to destruction of target tumor cells are partially understood. A population of cytolytic T cells have been identified which carry the CD8+ antigenic determinant on their surfaces. These cells require CD4+ helper lymphocytes for activation, which is a complex event mediated by antigen processing and presentation in association with the major histocompatibility complexes. Antigen processing assures that only cells targeted to the tumor antigens will be activated.
Monoclonal antibodies directed to various markers on subpopulations of T lymphocytes have been used to activate immune effector cells. OKT3 antibody administered by injection, for example, meets with CD3, and can cause a whole array of immune effects including IL-2, TNF-alpha, and IL-6 release, tissue damage, and either activation or suppression of T cell activity. More recently, OKT3 specificity has been combined with a antitumor specificity in a bispecific antibody. Link, et al., Blood, 81: 3343 (1993) showed that a bispecific antibody having one arm of OKT3 and the other arm directed to a B-cell malignant antigen was able to induce cytotoxicity of target tumor cells. Interestingly, the T-cell activation was without regard to the natural specificity of the T cell, and required the presence of the tumor cells. Thus, in the simultaneous binding of tumor cell and effector cell by the same antibody, the T cells are effectively recruited from the general T cell population, and retargeted to destroy the tumor cells.
It has also been shown by Weiner, Int. J. Cancer, Supplement 7, 63 (1992) that the action of the bispecific antibody is enhanced by coadministration of IL-2, so that combinational therapy resulted in management of a 100 to 1000 times greater tumor load than with the anti-tumor monoclonal antibody alone. Alternatively, the co-stimulus observed in the use of the bispecific antibody may be provided through binding of the Fc domain of the antibody to the Fc monocyte receptor, which in turn provide the co-stimulus, possibly through binding of the B7 family of membrane proteins to CD28. Preactivation ex vivo of cytotoxic T cells with co-administration of bispecific F(abxe2x80x2) has also been reported (Mezzanzanice, et al., Cancer Res., 51:5716 (1991).
The present invention utilizes a selection method designed to obtain monoclonal antibodies of very high specificity to particular tumor antigens, or to antigenic determinants shared by a single differentiated tissue type. It is also an objective to produce monoclonal antibodies which favor an externally disposed, stable, nonshed antigen. One problem with monoclonal antibodies raised to cell extracts derived from and screened against only one tumor or cell line, is their tendency to cross-react, however slightly, with vital non-tumor tissues. Many of the side-effects observed in immunotherapy are attributable to the nonspecific interaction of the antibody or antibody-toxin/drug/radionuclide conjugate with normal non-target cells, causing an associated toxicity. It is therefore an important object of this invention to provide an antibody of sufficient specificity that side-effects are minimized and limited to minor symptoms, or avoided.
Thus, in selecting an antibody specific for a common antigenic determinant displayed on the cell surface of cancers of a defined cell type, a mixture of cells is prepared, the mixture comprising cells from individual cell lines derived from a plurality of cancer cells of defined tissue type. This mixture of whole cells is then injected into a laboratory animal such as a mouse, according to a conventional immunization protocol to immunize the animal with the heterologous human tumor cells. Reactive B cells are then harvested from the animal, preferably the disrupted spleens, and fused with myeloma cells to form hybridomas. By maintaining the cell density below a critical level in which a statistical distribution function predicts one or two hybridomas per well, the likelihood of obtaining isolated single hybridomas was improved.
After cloning and outgrowth, supernatant medium containing the secreted monoclonal antibodies was removed. The screenings were then carried out, first, by contacting the mixture of cancer cells of the defined tissue, and a mixture of cancer cells of a different tissue type, with the monoclonal antibody under conditions conducive to binding of the antibody to cells displaying the target antigenic determinant. A fluorescent dye that recognizes the antibody is then added and the cells are then evaluated in a flow cytometer to determine which cells have detectable dye and which do not. The cell types are distinguished by a log scale of emission light intensity. Thus, the cells are ranked into a first class having labelled antibody bound to the surfaces thereof and into a second class having no labelled antibody bound, thereby showing a bimodal distribution of cells in flow cytometry.
The second screen involves further screening tests on the cells showing a bimodal distribution in which individual cells of prostate cancer and other cancer origin are labelled with the monoclonal antibody. Thus, each cancer cell type is individually tested with the labelled antibody to identify antibody with binding specificity for the cancer cells derived from the tissue of interest. Those antibodies which demonstrate unambiguous reactivity with prostate cancer-derived cells and no reactivity with nonprostate-derived cells are further tested. The cancer tissue types for which this method is intended in its therapeutic application include all those derived or arising from body organs unessential for viability such as ovary, breast, certain endocrine glands (thyroid), testicle, as well as prostate.
The third screening test is performed upon the monoclonal antibodies passing both the first and second screen, and involves determining the binding specificity of the labelled antibody for tissue sections derived from a plurality of cancers of defined cell type, together with controls of normal tissue sections from nonhomologous tissue.
In a variation of the above method involving the production of monoclonal antibodies specific for prostate tissue, the steps set forth therein are reproduced, with the exception that the initial immunization is carried out by preparing a whole cell suspended mixture of cells from cell lines derived from a plurality of prostatic tumors, proceeding through the next steps to grow out the hybridomas, but then performing the first screening test upon a mixture of the tumor cells contained in the inoculum and cells from cell lines not of prostate origin, after incubation with the labelled antibody. This variation in procedure does not screen against monoclonal antibodies reacting with both normal prostate and prostatic tumor cells, while not recognizing the nonprostate cells.
The invention herein encompasses the monoclonal antibodies obtained by the above disclosed methods, and also various diagnostic and therapeutic reagents made from them. Particularly useful are antibody conjugates in which a reporter molecule or detectable label is chemically conjugated to the antibody or an antibody fragment which retains the antigen binding characteristics and specificity of the originally isolated antibody. These reporters may be direct, such as a radioactive isotope or dye, or indirect, such as an enzyme, where the enzyme subsequently acts on a substrate to produce a detectable signal. In diagnostics, the reagents of the present invention will have especial efficacy in evaluating histologic sections and in immunoscintigraphy.
In therapeutic application in which the eradication of tumor cells is sought, the same antibodies will be efficacious, but with different conjugated moieties which are adapted for inducing cell death of the tumor cell to which they bind. The cytotoxic moieties may include a radionuclide, a chemotherapeutic drug, or a biologic toxin. In diagnostics application, tumor cells of unessential organs or tissue types widely distributed in the body may be used in the immunization, and then screened according to the present method. The monoclonals obtained cannot be used for therapeutic purposes, since normal tissue of the same type would be destroyed. However, they are useful as reagents in diagnostics, as in the tissue section illustrated in FIG. 4.
Applicants have also found that a bispecific construct which binds two antigens has cytotoxic activity against a tumor cell in the presence of one or more effector cells. The construct contains one or more polypeptides and is characterized in having a first binding domain having binding specificity for an antigenic determinant expressed on the surface of malignant and normal cells of defined nonessential animal tissue type, and a second binding domain having binding specificity for an antigenic determinant expressed on the surface of a defined class of immune effector cells such as monocyles, T-cells or NK cells. Antigenic determinants of tissue specific type include, for example, the determinant recognized by the 5E10 monoclonal antibody specific for prostate tissue. Other nonessential tissues having tissue specific determinants for which monoclonal antibodies may be isolated by the method of the present invention, include the thymus, thyroid, mammary gland, testes, ovaries, etc. Monoclonal antibodies specific for lymphocyte markers may be directed against CD3 (such as the commercially available OKT3), CD4, CD8, CD16, and CD28, CD32 and CD64.
Finally, in a still further embodiment of the present invention, a hybrid-hybridoma cell line is provided by first fusing an OKT3 secreting cell (ATCC Accession No. CRL 8001) with a 5E10 prostate tissue specific monoclonal antibody secreting cell, and growing out the fusion hybrid in a selective medium. The resulting hybrid-hybridoma cell line secretes monoclonal OKT3 antibody, 5E10 antibody, and a bispecific antibody having the combined specificity of OKT3 on one antibody arm, and 5E10 specificity on the other antibody arm.